THE OSMOTIC BEHAVIOR OF ROD PHOTORECEPTOR OUTER SEGMENT DISCS

The permeability properties of frog rod photoreceptor outer segment discs were investigated in preparations of purified, dark-adapted, outer segment fragments by the techniques of direct volume measurement and electron microscopy. Outer segment discs were found to swell and contract reversibly in response to changes in the osmotic pressure of the bathing medium in accordance with the Boyle-van't Hoff law. By use of the criterion of reversible osmotic swelling, the disc membrane is impermeable to Na⁺, K⁺, Mg⁺², Ca⁺², Cl^-, and (PO₄)^-3 ions, whereas it is freely permeable to ammonium acetate. The disc membrane is impermeable to sucrose, although its osmotic behavior towards this substance is different from its behavior towards impermeable ions. Electron microscopy showed that the osmotic effects on the rod outer segment fragments represent changes in the intradiscal volume. Fixation with glutaraldehyde did not abolish the permeability properties of the disc membrane, and fixed membranes were still capable of osmotic volume changes. It is concluded from this study that the frog's rod photoreceptor outer segment discs are free-floating membranous organelles with an inside space separate and distinct from the photoreceptor intracellular space.     

MEMBRANE CHARACTERISTICS AND OSMOTIC BEHAVIOR OF ISOLATED ROD OUTER SEGMENTS

Freshly isolated frog rod outer segments are sensitive osmometers which retain their photosensitivity; their osmotic behavior reveals essentially the same light-sensitive Na⁺ influx observed electrophysiologically in the intact receptor cell. Using appropriate osmotic conditions we have examined freeze-etch replicas of freshly isolated outer segments to identify the membrane which regulates the flow of water and ions. Under isosmotic conditions we find that the disc to disc repeat distance is almost exactly twice the thickness of a disc. This ratio appears to be the same in a variety of vertebrate rod outer segments and can be reliably measured in freeze-etch images. Under all our osmotic conditions the discs appear nearly collapsed. However, when the length of the outer segment is reduced by hyperosmotic shocks the discs move closer together. This markedly reduces the ratio of repeat distance to disc thickness since disc thickness remains essentially constant. Thus, the length reduction of isolated outer segments after hyperosmotic shocks primarily results from reduction of the extradisc volume. Since the discs are free floating and since they undergo negligibly small changes in volume, the plasma membrane alone must be primarily responsible for regulating the water flux and the light-sensitive Na⁺ influx in freshly isolated outer segments. On this basis we calculate, from the osmotic behavior, that the plasma membrane of frog rod outer segment has a Na⁺ permeability constant of about 2.8 x 10^-6 cm/s and an osmotic permeability coefficient of greater than 2 x 10^-3 cm/s.     

Light sensitive zinc content of protein fractions from bovine rod outer segments

Bovine retinas, isolated rod outer segments and emulphogene extracts of rod outer segments have been shown to contain appreciable amounts of Zn²⁺, Ca²⁺ and Mg²⁺ when isolated in the absence of added metal ions. Chromatography of emulphogene extracted rod outer segments in Sephadex G-25 showed virtually all the Ca²⁺, Zn²⁺ and protein to elute with the void volume. Levels of Zn²⁺ but not Ca²⁺ were light sensitive. The Zn²⁺ contents of protein fractions were about 60% higher when samples were bleached. Under optimal conditions protein fractions contained 1.4 – 1.8 g atoms Zn²⁺/mole rhodopsin for dark adapted samples and 2.1 to 3.2 g atoms Zn²⁺/mole of rhodopsin for bleached samples.     

Biochemical aspects of the visual process XXXII. Movement of sodium ions through bilayers composed of retinal and rod outer segment lipids

The leakage of Na⁺ from sonicated liposomes, composed of rod outer segment lipids, retinal lipids and a 4 : 1 phosphatidylcholine/phosphatidylserine mixture, has been studied. Both retinal and rod outer segment lipid liposomes lose Na⁺ faster than Ca²⁺ which indicates that the observed leakage occurs from closed liposomal structures.Liposomes from rod outer segment lipids are extremely leaky, losing sodium about 10 times as fast as retinal lipid liposomes and twice as fast as the phosphatidylcholine/phosphatidylserine liposomes.This high permeability of rod outer segment lipid liposomes, as compared to retinal lipid liposomes, is probably due to both the higher degree of unsaturation of the fatty acid chains and their lower cholesterol content. In the rod outer segment lipid extract 48% of the fatty acid chains consists of docosahexaenoic acid (C_22:6) against only 24% in retinal lipid extract. Rod outer segment lipids contain 4.0% cholesterol against 12.3% in retinal lipids.The sodium leakage from rod outer segment lipid liposomes is little affected by the presence of 5 mM calcium in the external dialysis medium, but with the two other types of liposomes significant decreases in permeability of about 20% are observed.The results are discussed in connection with the role of cations in visual excitation.     

Control of sodium influx by calcium and turgor in two charophytes differing in salinity tolerance

The effects of Ca²⁺ and cell turgor on Na⁺ influx were examined in two charophytes, lamprothamnium papulo-SUM (salt-tolerant) and Chara corallina (salt-sensitive), to try to identify causes of salinity toxicity. Mortality was associated with Na+ influx, with the two species showing similar sensitivities to high Na⁺ influx. In Lamprothamnium, toxic influxes of Na⁺ occurred at much higher external Na⁺ concentrations than in Chara. The differences in Na⁺ influx at the same Na⁺ concentration were not due to different responses to external Ca²⁺. Lamprothamnium adjusts its turgor in response to increasing NaCl whereas Chara cannot. In solutions of KC1 up to at least 200 mol m_-3, however, Chara regulated turgor, and when KC1 was subsequently replaced with NaCl, Na+ influx was low and similar to that in Lamprothamnium at the same Na* concentration. Chara cells which were not turgor-adjusted in KCI had Na⁺ influxes 2-5-fold higher than the turgid cells. Thus, it appears that turgor is a major determinant of Na⁺ influx, and therefore of cell survival. We found no evidence that the mechanism of Na⁺ influx in Chara is different from that in Lamprothamnium. Higher susceptibility of Chara to NaCl seems to result from inability to regulate turgor, in turn leading to toxic Na⁺ influx.     

Ouabain switches Na+ transport to Na+/Na+ equivalent exchange in normal and apoptotic lymphoid cells

A theory of change of the ionic fluxes in the lymphoid cells in their transition from normal to apoptosis we have developed previously is applied to the analysis of Na⁺/Na⁺ exchange fluxes in human lymphoid cells U937 exposed to ouabain. We solve a system of equations describing changes in the intracellular concentrations of Na⁺, K⁺ and Cl^?, membrane potential and cell volume. It is shown that the Na⁺ influx (I _Na/Na) and output flux through the Na⁺/Na⁺ tract increased 4 times in 8 h after disconnecting Na⁺/K⁺-ATPase for normal cell U937. These fluxes increased 2.6 times for apoptotic cells. The value of I _Na/Na after 8 h off pump by ouabain is 97% of the total Na⁺ input for both cell types. It is concluded that ouabain not only inhibits the Na⁺/K⁺-ATPase, but also increases Na⁺ exchange fluxes through the Na⁺/Na⁺ tract, thereby switching sodium transport across the membrane of lymphoid cells to Na⁺/Na⁺ equivalent exchange.     

Interphotoreceptor retinoid-binding protein is the physiologically relevant carrier that removes retinol from rod photoreceptor outer segments

Light detection by vertebrate rod photoreceptor outer segments results in the destruction of the visual pigment, rhodopsin, as its retinyl moiety is photoisomerized from 11-cis to all-trans. The regeneration of rhodopsin is necessary for vision and begins with the release of the all-trans retinal and its reduction to all-trans retinol. Retinol is then transported out of the rod outer segment for further processing. We used fluorescence imaging to monitor retinol fluorescence and quantify the kinetics of its formation and clearance after rhodopsin bleaching in the outer segments of living isolated frog (Rana pipiens) rod photoreceptors. We independently measured the release of all-trans retinal from bleached rhodopsin in frog rod outer segment membranes and the rate of all-trans retinol removal by the lipophilic carriers interphotoreceptor retinoid binding protein (IRBP) and serum albumin. We find that the kinetics of all-trans retinol formation in frog rod outer segments after rhodopsin bleaching are to a good first approximation determined by the kinetics of all-trans retinal release from the bleached pigment. For the physiological concentrations of carriers, the rate of retinol removal from the outer segment is determined by IRBP concentration, whereas the effect of serum albumin is negligible. The results indicate the presence of a specific interaction between IRBP and the rod outer segment, probably mediated by a receptor. The effect of different concentrations of IRBP on the rate of retinol removal shows no cooperativity and has an EC50 of 40 micromol/L.     

On the mechanisms of sodium ion transport by the irrigated gills of rainbow trout (Salmo gairdneri)

Sodium uptake by rainbow trout gills has been investigated with a small-volume system enabling rapid, successive flux measurements in different solutions. Sodium influx obeys a Michaelis-Menten type relation, with a K_m of 0.46 mM, and uptake proceeds unimpaired in the absence of penetrating counter-ions. This suggests a coupled cation exchange. Ammonia output is about the same as the Na⁺ influx when external [Na⁺] is 1 mM, but at higher or lower Na⁺ influxes, the correlation does not hold. A progressive downward shift in the pH of the irrigating medium as Na⁺ influx increases indicates that the exchanging cation is hydrogen. In support of this, acetazolamide, which inhibits Na⁺ uptake, also prevents the downward pH shift. The potential across the gill is about 10 mv, body fluids positive, in NaCl solutions up to 10 mM, and is little affected by changes in Na⁺ concentration below that. Finally, evidence for locating the rate-limiting step at the outer membrane of the epithelium is presented.     

Ouabain-sensitive 86Rb(K) influx is linked to transepithelial Na transport in pig kidney cell line

The pig kidney cell line, LLC-PK₁, exhibits rheogenic d-glucose coupled transepithelial Na⁺ transport that is inhibited by phlorizin. By measuring the difference in initial rates of influx of ⁸⁶Rb⁺ with and without coupled Na⁺ transport, we can demonstrate an ⁸⁶Rb⁺ uptake linked to Na⁺ transport. The simultaneous determination of phlorizin-inhibited Na coupled d-[³H]glucose uptake and ⁸⁶Rb⁺ influx allows calculation of an Na⁺/Rb⁺ stoichiometry that is consistent with an electrogenic Na⁺ for Rb⁺ exchange.     

Sodium transport in red blood cells of lamprey LAmpetra fluviatilis

• 1.1. Unidirectional Na⁺ influx in lamprey red blood cells was determined using ²²Na as a tracer.
• 2.2. Total Na⁺ uptake and amiloride-inhibitable Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration (Na_e).
• 3.3. At 141 mM Na_e, the average value of net Na⁺ influx was 13 ± 1.1 and the amiloride-sensitive Na⁺ influx was 5.3±1.1 mmol/l cells per hr (±SE).
• 4.4. The amiloride-sensitive component of Na⁺ influx was significantly activated by 10⁻⁵ M isoproterenol, by 2 × 10⁻⁵ M DNP, and by cell shrinkage.
• 5.5. Furosemide (1 mM) had no effect on the Na⁺ transport in red cells.
• 6.6. The residual amiloride-insensitive component of Na⁺ transport was a linear function of Na_e in the range of 5–141 mM. This transport seems to be accounted for by simple diffusion.

     

Na movements and their oscillations during fertilization and the cell cycle in sea urchin eggs

We have analysed in detail the Na⁺ content and Na⁺ influx during fertilization and first divisions of the sea urchin egg (Paracentrotus lividus) using a filtration technique devised to eliminate rapidly contamination by the Na⁺ of external sea water. In the first 5 min following fertilization the egg fills up with Na⁺ (+ 30%). Thereafter Na⁺ is extruded and the Na⁺ content stabilizes at about 60% of the unfertilized egg level by the second cleavage (2 h). The initial increase in Na⁺ content is due to a large increase in Na⁺ influx already detected at 20 sec. The Na⁺ influx reaches its maximum at 1 min and its minimum at 5 min. H⁺ excretion follows the same kinetics. A second increase in Na⁺ influx is noted 5–10 min after fertilization; it reaches its maximum at prophase metaphase (30 min) and its minimum during cleavage (60 min). These oscillations in Na⁺ influx were observed for the first three divisions. Fertilization also immediately stimulates the Na⁺ efflux which remains elevated throughout the cell cycle and is responsible for the depletion of the Na⁺ content of the embryos. Activation of the eggs by weak amine bases (5 mM NH₄Cl) which bypasses the early cortical reaction produces only a depletion in the Na⁺ content of the egg similar to that produced by fertilization. NH₄Cl also increases the Na⁺ influx soon after fertilization, although no transient variations are noted.     

Measurement of fast light-induced disc shrinkage within bovine rod outer segments by means of a light-scattering transient

A fast light-induced light-scattering transient, previously found in rod outer segment suspension, the so-called P-signal (Hofmann, K.P., Uhl, R., Hoffmann, W. and Kreutz, W. (1976) Biophys. Struct. Mechanism 2, 61–77), is described in more detail.The effect has the same action spectrum as rhodopsin bleaching. It is not regenerated with 11-cis retinal.The response is not linear with light-intensity for flashes which bleach more than 2.0% of rhodopsin; it saturates at an intensity corresponding to 15% rhodopsin bleaching.The wavelength- and scattering angle dependence lead to the conclusion that the change in light-scattering reflects a shrinkage of an osmotic compartment of the rod outer segment.The only compartment which we found to be intact in our rod outer segment preparations was the disc or rod sac; therefore, the effect must be attributed to a light-induced shrinkage of the rhodopsin-containing disc organelles.The overall effect (15% of rhodopsin is bleached) is in the range of 0.5–1.5% of the original volume.A light-induced passive cation-efflux from the disc, e.g. of Ca²⁺, can be ruled out as a possible molecular origin of the disc-shrinkage in our preparations.     

Actions of Ca2+ on an Early Stage in Phototransduction Revealed by the Dynamic Fall in Ca2+ Concentration during the Bright Flash Response

To study the actions of Ca²⁺ on “early” stages of the transduction cascade, changes in cytoplasmic calcium concentration (Ca²⁺ _i) were opposed by manipulating Ca²⁺ fluxes across the rod outer segment membrane immediately following a bright flash. If the outer segment was exposed to 0 Ca²⁺/0 Na⁺ solution for a brief period immediately after the flash, then the period of response saturation was prolonged in comparison with that in Ringer solution. But if the exposure to 0 Ca²⁺/0 Na⁺ solution instead came before or was delayed until 1 s after the flash then it had little effect. The degree of response prolongation increased with the duration of the exposure to 0 Ca²⁺/0 Na⁺ solution, revealing a time constant of 0.49 ± 0.03 s. By the time the response begins to recover from saturation, Ca²⁺ _i seems likely to have fallen to a similar level in each case. Therefore the prolongation of the response when Ca²⁺ _i was prevented from changing immediately after the flash seems likely to reflect the abolition of actions of the usual dynamic fall in Ca²⁺ _i on an early stage in the transduction cascade at a site which is available for only a brief period after the flash. One possibility is that the observed time constant corresponds to the phosphorylation of photoisomerized rhodopsin.     

Bumetanide-sensitive Ion Fluxes in Vascular Smooth Muscle Cells: Lack of Functional Na+, K+, 2 Cl− Cotransport

To examine the involvement of Na⁺,K⁺,2Cl⁻ cotransport in monovalent ion fluxes in vascular smooth muscle cells (VSMC), we compared the effect of bumetanide on ⁸⁶Rb, ³⁶Cl and ²²Na uptake by quiescent cultures of VSMC from rat aorta. Under basal conditions, the values of bumetanide-sensitive (BS) inward and outward ⁸⁶Rb fluxes were not different. Bumetanide decreased basal ⁸⁶Rb uptake by 70–75% with a K _i of ∼0.2–0.3 μm. At concentrations ranging up to 1 μm, bumetanide did not affect ³⁶Cl influx and reduced it by 20–30% in the range from 3 to 100 μm. In contrast to ⁸⁶Rb and ³⁶Cl influx, bumetanide did not inhibit ²²Na uptake by VSMC. BS ⁸⁶Rb uptake was completely abolished in Na⁺- or Cl⁻-free media. In contrast to ⁸⁶Rb, basal BS ³⁶Cl influx was not affected by Na⁺ _o and K⁺ _o . Hyperosmotic and isosmotic shrinkage of VSMC increased ⁸⁶Rb and ³⁶Cl influx to the same extent. Shrinkage-induced increments of ⁸⁶Rb and ³⁶Cl uptake were completely abolished by bumetanide with a K _i or ∼0.3 μm. Shrinkage did not induce BS ⁸⁶Rb and ³⁶Cl influx in (Na⁺ or Cl⁻)- and (Na⁺ or K⁺)-depleted media, respectively. In the presence of an inhibitor of Na⁺/H⁺ exchange (EIPA), neither hyperosmotic nor isosmotic shrinkage activated ²²Na influx. Bumetanide (1 μm) did not modify basal VSMC volume and intracellular content of sodium, potassium and chloride but abolished the regulatory volume increase in isosmotically-shrunken VSMC. These data demonstrate the absence of the functional Na⁺,K⁺,2Cl⁻ cotransporter in VSMC and suggest that in these cells basal and shrinkage-induced BS K⁺ influx is mediated by (Na⁺ _o + Cl⁻ _o )-dependent K⁺/K⁺ exchange and Na⁺ _o -dependent K⁺,Cl⁻ cotransport, respectively. Received: 30 January 1996/Revised: 20 May 1996     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

Activation of phosphodiesterase in frog rod outer segment by an intermediate of rhodopsin photolysis. I.

Guanosine 3′,5′-cyclic monophosphate phosphodiesterase (EC 3.1.4.1) in frog rod outer segment prepared by a sucrose stepwise density gradient method was activated by light in the presence of GTP. Rhodopsin in rod outer segment was solubilized with sucrose laurylmonoester and then purified by concavanalin A-Sepharose column. Addition of photo-bleached preparation of the purified rhodopsin to the rod outer segment, which had been prepared by 43% (w/w) sucrose floatation, caused the activation of phosphodiesterase in the dark, while each component of the photo-product eluted from the column (all-trans retinal and opsin) did not. Regenerated rhodopsin prepared from 11-cis retinal and purified opsin activated phosphosdiesterase when it was bleached. From these facts it is suggested that an intermediate or a process of photolysis of rhodopsin causes activation of phosphodiesterase.     

Controls on na influx in corn roots

We have investigated the effects of hyperpolarization and depolarization, and the presence of K⁺ and/or Ca²⁺, on ²²Na⁺ influx into corn (Zea mays L.) root segments. In freshly excised root tissue which is injured, Na⁺ influx is unaffected by hyperpolarization with fusicoccin, or depolarization with uncoupler (protonophore), or by addition of K⁺. However, added Ca²⁺ suppresses Na⁺ influx by 60%. In washed tissue which has recovered, Na⁺ influx is doubled over that of freshly excised tissue, and the influx is increased by fusicoccin and suppressed by uncoupler. This energy-linked component of Na⁺ influx is completely eliminated by low concentrations of K⁺, leaving the same level and kind of Na⁺ influx seen in freshly excised roots. The K⁺-sensitive energy linkage appears to be by the carrier for active K⁺ influx. Calcium is equally inhibitory to Na⁺ influx in washed as in fresh tissue. Other divalent cations are only slightly less effective. Net Na⁺ uptake was about 25% of ²²Na⁺ influx, but proportionately the response to K⁺ and Ca²⁺ was about the same.

The constancy of K⁺-insensitive Na⁺ influx under conditions known to hyperpolarize and depolarize suggests that if Na⁺ transport is by means of a voltage-sensitive channel, the rise or fall of channel resistance must be proportional to the rise or fall in potential difference. The alternative is a passive electroneutral exchange of ²²Na⁺ for endogenous Na⁺. The data suggest that an inwardly directed Na⁺ current is largely offset by an efflux current, giving both a small net uptake and isotopic exchange.

     

Light-enhanced cross-linking of rhodopsin in rod outer segment membranes as detected by chemical probes

Bovine rod outer segment membranes were treated with cross-linking reagents before and after light exposure. Bleached membranes showed enhanced cross-linking with difluorodinitrobenzene or methyl acetimidate compared to dark-adapted membranes. The light-induced enhancement of cross-linking may be due to increased association of rhodopsin monomers in the light and/or due to increased reactivity of amino and sulfhydryl groups of bleached rhodopsin. In some instances, the band ascribed to the rhodopsin monomer in gel electrophoresis appears as a partially resolved doublet. Treatment of bleached rod outer segment membranes with methyl acetimidate improved the resolution of the doublet into two closely migrating bands.     

Sodium transport in erythrocytes of the mollusc Anadara broughtonii: Evidence for inhibition by quinine

Sodium transport through the molluscan erythrocyte membrane was examined using ²²Na as a tracer. Incubation of the red cells in standard saline resulted in a rapid ²²Na uptake reaching steady state concentration (about 21.5 mmol/l cells) in the first 60 min. A similar pattern in the time course of ²²Na uptake was seen in the erythrocytes incubated in mantle fluid. The average value of unidirectional Na⁺ influx, measured as a 5-min ²²Na uptake, was 7.76 ± 0.36 mmol/1 cells/5 min or 93 ± 4.3 mmol/1 cells/hr. The initial rate of Na⁺ influx increased in a saturable fashion as a function of external Na⁺ concentration with apparent AT., of 380±12mM and V_max of 14.3 ± 2.4 mmol/1 cells/5 min. Amiloride (1 mM), furosemide (1 mM), and DIDS (0.1 mM) had no effect on either initial Na⁺ influx (5 min ²²Na uptake) or equilibrium Na⁺ concentration (60 min and 120min ²²Na uptake) in the molluscan red cells exposed to standard saline. Quinine (1 mM) caused a significant fall in the initial Na⁺ influx (by 48%) and in 60-min ²²Na uptake (by 32%) as compared with control levels. In the presence of 0.1 mM ouabain, ²²Na uptake into the red cells was enhanced by an average 27% and 44% during 60 min and 120 min of cell incubation, respectively. The ouabain-sensitive Na⁺ accumulation in the red cells reflected a contribution of the Na, K-pump to Na⁺ transport and the mean value was 5.6 ± 1.0 mmol/1 cells/hr.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.